HIV/AIDS is a global pandemic with 37 million individuals living with HIV infection and approximately 39 million have died from AIDS worldwide. The objectives of this project are to define the unique epidemiological, clinical, virologic, and immunologic features of HIV and its co-infections in developing countries, to determine the viral kinetics associated with sexual transmission, and to characterize the molecular strains of HIV internationally for infectiousness and progression of disease. We continued our research on the development, validation and application of accurate HIV cross-sectional incidence testing. We compared serologic characteristics of young adults with perinatally-acquired HIV infection to two groups of individuals who were infected as adults: elite controllers and adults exposed to ART. Young adults who acquired HIV perinatally comprise a significant proportion of HIV-infected individuals in some epidemics. The incidence assays evaluated included the limiting antigen avidity assay (LAg-Avidity) and the BioRad avidity assay. We tested 225 samples (66 which were from virologically detectable time points) from 22 perinatally infected young adults who had been infected a minimum of 13 years. We compared these results to samples from elite controllers (21 subjects, 40 samples) and ART exposed individuals (317 subjects, 480 samples of which 190 had detectable viral loads). For the latter two groups, all had been infected as adults for at least 2 years and some had been on ART more than 2 years. Half (11/22) of the perinatally infected young adults had low LAg-Avidity values that would misclassify as recent infection, compared to 7% (27/379) of ART treated subjects and 20% (4/21) of elite controllers. For the BioRad avidity assay, 38% (7/22) of the perinatally infected young adults had low LAg-Avidity values that would misclassify as recent infection, compared to 0.8% (3/379) of ART treated subjects and 0% (0/21) of elite controllers. Young adults infected perinatally were more likely to be misclassified as recently-infected by incidence assays than their counterparts who were infected as adults. These results would falsely increase HIV incidence estimates in studies using these assays in populations that include perinatally-infected adults. The use of HIV sequence data to identify clusters of individuals infected with related viruses is important for assessing epidemiological and evolutionary dynamics in populations and is an important tool to both design and evaluate control strategies. We compared two approaches used to identify clusters of individuals with a shared HIV infection history: Cluster Picker and HIV-TRACE. We used three gp41 datasets from the Rakai Community Cohort Study: 1) next-generation sequences (NGS) from nine linked couples; 2) NGS from longitudinal sampling of 14 patients; and 3) consensus sequences from a cross-sectional dataset (n=1022) containing 91 cohabitating heterosexual couples. We calculated the optimal genetic distance threshold to separate linked versus unlinked NGS datasets using a receiver operating curve analysis. We evaluated the number, size, and composition of clusters detected by Cluster Picker and HIV-TRACE at six genetic distance thresholds (1%5.3%) on all three datasets. The optimal genetic distance threshold for this population using this fragment of the HIV genome to distinguish linked and unlinked couples was 5.3% with an ROC of > 99%. HIV-TRACE tended to detect larger clusters, whereas Cluster Picker detected more clusters containing only two sequences. At the lower cut off value of 2%, 55% (50/91) cohabitating couples did not cluster at all, while at the optimal cut off for this population, 80% (73/91) couples were molecularly linked. These results demonstrate that definitions of viral clusters for HIV vary depending on the population assessed and the research question posed. The major barrier to curing HIV infection is the persistence of HIV in latently infected resting memory CD4+ T cells. Previous studies have quantified this pool of latently infected cells in North Americans and Europeans but no study had quantitated the latent HIV-1 reservoir (LVR) in sub-Saharan Africans, who make up the largest population of HIV-infected individuals globally. We therefore conducted a study in 70 virally suppressed HIV-infected individuals in Rakai, Uganda to determine the minimum frequency of latently infected cells with replication competent virus using the quantitative viral outgrowth assay (Q-VOA). The median frequency of latently infected rCD4 cells in this Ugandan cohort was 0.36 infectious units per million cells (IUPM; 95%CI=0.26-0.55 IUPM), three-fold lower than the frequency observed in the Baltimore cohort (1.08 IUPM; 95%CI=0.72-1.49 IUPM; p<0.001). Reservoir size in Ugandans correlated positively with set-point viral load and negatively with duration of viral suppression. Preliminary analysis of viral subtype suggests no significant difference in LVR size between individuals infected with subtype A vs. subtype D. Of note, the LVR size in this Ugandan population is over 3-fold smaller than that previously reported in Americans. This represents the first quantification of latently infected resting CD4+ T cells with replication competent virus in an ART treated, virally suppressed sub-Saharan African population. Further immunologic correlates and decay over time analyses are ongoing. The Johns Hopkins Emergency Department has served as an observational window on the HIV-epidemic in inner city Baltimore for over 25 years. We recently analyzed 7 discrete identity-unlinked serosurveys conducted on 18,240 untargeted adult JHH-ED patients between 1987-2013 for demographic trends in HIV prevalence, cross-sectional incidence estimates, viral load and HCV prevalence. HIV prevalence in 1987 was 5.2%, peaked at 11% from 1992-2003, and then declined to 5.6% in 2013. Proportion of undiagnosed HIV declined over time from 77% in 1987 to 12% by 2013. HIV incidence estimates in 2001 were 2.1% and declined steadily to 0.16% by 2013. Proportion of HIV+ individuals with viral suppression increased steadily from 23% in 2001 to 59% by 2013. Consistent with increasing viral suppression, 80% of 214 HIV+ individuals surveyed in 2013 had antiretroviral drugs detected in their sera, a marked increase from 2007 (27%). However, HCV in this population remained at 18-19% from 1988 until 2007 and declined only slightly to 14% by 2013. We have expanded our use of ultra-deep pyrosequencing to identify HIV superinfection and describe its effects on the pandemic. In addition, we have utilized this same technology to examine other aspects of HIV disease. In particular, we assisted in the viral linkage determination for transmission cases in the HPTN 052 clinical trial, which demonstrated that fully suppressive ART can drastically decrease HIV transmission in discordant couples. We also used this technology to verify resistance data from the HPTN 068 pre-exposure prophylaxis trial, and a study of ART resistance in HIV infected Cameroonians. We have expanded our collaborations to examine the role of immune activation, and increased cytokine levels on herpetic reactivation after the initiation of ART. We found that ART initiation increases levels of IL-6, which was associated with HSV-2 and CMV shedding in women from Rakai Uganda. This finding will be examined in more detail in a larger population in Uganda and at the NIH clinical center.